FALL 2013Week 13 & 14: 11/18/2013 - 12/1/2013 Summary of Week 13 & 14: Another sample of PfFabG was expressed, purified, and characterized. However, when the SDS-Page gel was analyzed, there was little to no band at the correct location (~33 kDa) on the gel which means that expression was likely unsuccessful. More virtual screening was conducted and the data was combined together in an excel document.
Fig: Concentration analysis of the wash step during purification
Fig: Concentration analysis of Elution 1 Trial 1 during purification of PfFabG
Fig: Concentration analysis of the first elution of PfFabG of purification - Trial 2
Fig: Concentration analysis of the second elution of PfFabG during purification
Fig: Concentration analysis of PfFabG after elution 2 (Trial 2) during purification
When the samples (Elution 1, Elution 2, Concentrated Sample) were analyzed using the Nanodrop, the conentrations determined were very low. This provides more evidence that the protein did not expressed or was in the -80 degree celsius freezer for too long to be viable.
Fig: SDS-Page gel analysis of PfFabG
From left to right: ColorPlus Protein Ladder 0: Before IPTG Induction 2: Soluble Fraction 3: Flowthrough 4: Wash 5: Elution 1 6: Elution 2
PfFabG is supposed to elute around the 33 kDa mark. From the gel you can see that the protein appears up until the wash sample and is missing in Elution 1 and 2. Based on the previous gel, I do not believe that there is a problem with the His-tag adhering to the Ni ions because there was a band at 33kDa in Elution 1 and 2 in the previous gel. There may have been a problem with purification in general. I used the same purification column as before so there could be an issue with the the age of the column or the incubation step. Week 11 & 12: 11/4/13 - 11/17/2013 Good--nice job on the protein -UMSummary of Week 11 & 12: GOLD docking of the CB306 library was conducted and analyzed. An enzyme assay was conducted on the enzyme collected two weeks ago to test for enzyme functionality. Due to the results collected from the time based trial, it appears that the protein is indeed functional.
Table 1: Gold docking analysis of CB306 compound library during the initial screening. The first 33 compounds will be further analyzed for more thorough analysis.
Ligand name
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
'5761107'
66.83
-59.11
2.68
0
0
0
0.18
0.02
'7722615'
67.72
-62.47
1.99
0
0
0
0.52
0.13
'7757183'
65.44
-55.07
3.74
0
0
0
1.55
2.23
'9027311'
65.13
-57.29
1
2
0
0
0.62
0.08
'7575772'
64.69
-49.8
5.55
0
0
0.5
0.74
0.21
'7676009'
64.5
-56.59
4
0
0
0
3.11
2.09
'7938694'
63.67
-61.61
1
1
0
0
2.04
0.1
'7842136'
62.75
-38.7
8.17
0
0
0
0.28
0.1
'5531766'
62.69
-55.43
3.11
0
0
0
2.21
2.36
'7558664'
62.53
-55.08
3.26
0
0
0
1.55
0.77
Table 2: Gold docking scores of the second run of the top 33 compounds in the CB306 library. The top four scores/10% of the second run are shown.
Rank
Ligand name
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
1
7558664
70.73
-65.45
3.13
0
0
0
2.36
0.61
2
6629394
70.59
-49.11
6.41
1
0
0.01
1.57
2.41
3
7938694
70.01
-64.31
2
1
0
0
1.81
0.27
4
7575772
69.45
-51.76
6.75
0
0
0
1.31
0.07
During the second run of the CB306 docking, the top ligands that were determined in the previous Run 2 were not ranked as highly in the second run. There is however a low standard deviation between each score. Although the scores may have ranked differently, the GOLD score between each ligand does not change by a large percentage.
Fig: Enzyme assay UV-Vis spectrophotometer time-based reading. Light blue line - absorbance at 340 nm for Trial 1. Dark blue line - absorbance at 340 nm for Trial 2. Light red line - absorbance at 340 nm for Trial 1. Dark red line - absorbance at 260 nm for Trial 2.
Because there is a subtle decrease in the absorbance of NADPH at 340 nm, this may indicate that the protein is functional. For the light blue line, the peaks that appear in the line are times when more enyme was added to the measured sample. Week 9 & 10: 10/23/13 - 11/3/2013 Good work Melissa! Do you have your FPLC results that you could post on here? Also, it would be good to include analysis on your virtual screening results. - Suman 11/4/13 Summary of Week 9 & 10: Protein was expressed, purified, and characterized during week 9. The protein was loaded into the FPLC for further concentration. Then the protein was stored in 20% glycerol. Protein expression was re-conducted due to a low concentration in protein. For virtual screening, 2C07 was analyzed through MolProbity and control and CB306 docking was conducted. Then the DH5alpha cells from cloning during the summer were regrown on a master plate and midiprepped for a larger stock of plasmid concentration. Fig: Concentration analysis of PfFabG after protein purification. The average concentration after Elution 1 was 0.645 mg/mL. Fig 2: Concentration analysis of the concentrated elution 1 with an averge concentration of 1.59 mg/mL and an average 260/280 of 1.825. Fig 3: The average concentration of Elution 2 was 0.28 mg/ml with an average 260/280 value of 0.94. Fig: Concentration analysis of PfFabG after FPLC but before concentrating the sample. The average concentration is very low at 0.155 mg/mL. Overall, protein purification will have to be scaled up in order to collect a larger yield of protein during round 3 of expression. Molprobity AnalysisResolution: 1.5 angstromsClashscore: 11.95Poor rotamers: 0Ramachandran outliers: 0Ramachandran favored: 236Cbeta deviations >0.25 A: 0MolProbity score ^: 1.69Residues with bad bonds: 0/982Residues with bad angles: 1/1224Error in Active Site: Contacts, McSc contacts, Hets contacts, 1 angle Overall there are few errors in active site and the protein structure was modified with flips and without hydrogens. Table 1: Gold score table of control docking of PfFabG
Ligand name
Score
S(PLP)
S(hbond)
S(cho)
S(metal)
DE(clash)
DE(tors)
intcor
NADPH
'15983949_NADPHpos8'
103.54
-71.38
10.18
1.86
0
0
2.93
1.89
Catechin Gallate
'6419835_catechingallate'
81.46
-60.12
7.22
0
0
0
0.16
0.02
Epigallocatechin gallate
'65064_epigallocatechingallate'
77.64
-59.07
6.92
0
0
0
1.11
0.04
Myricetin
'5281672_myricetin'
64.71
-42.32
7.47
0
0
0
0.91
1.81
Chloroquine
'2719_chloroquine'
62.98
-61.23
0.84
0.4
0
0
1.07
0.15
Heptadecynoic Acid
'22600366_heptadecynoicacid'
61.67
-56.09
2.19
0
0
0
0.57
0.1
AG-690/10423014
'17399864_AG690'
60.74
-56.91
1
0.79
0
0
0.77
0
Morin
'5281670_morin'
59.68
-44.93
5.03
0
0
0
1.12
1.91
Luteolin
'5280445_luteolin'
58.41
-42.15
5.71
0
0
0
0.43
0
AC1NU03A
5442438_AC1NU03A'
55.91
-58.66
0
0
0
0.44
1.43
0.25
ZINC19166762
28082271_ZINC19166762'
53.49
-49.45
1.99
0
0
0
1.08
0.12
Hexachlorophene
3598_hexachlorophene'
51.17
-48.47
1
0
0
0
0.15
0
AI-204/31720014
'16654126_AI204'
50.23
-47.25
0.61
0.47
0
0
0.12
0
Chrysin
'5281607_chrysin'
49.85
-44.16
2
0
0
0
0.16
0
Aspirin
'2244_aspirin'
41.94
-36.39
1.95
0
0
0
0.54
0.78
Week 7 & 8: 10/07/2013 - 10/20/13Good work Melissa. Try to include a brief analysis of the Nanodrop data. Do you have any virtual data? Thank you. -Max 10/21/13
Summary of Week 7 & 8: SDS-PAGE gel was analysed. PfFabG was spun down in the concentrator, yielding an average concentration of 2.52 mg/mL. Protein expression was attempted but due to time constraints, it could not be achieved. More preparation towards virtual screening was conducted. The positive and negative control ligands were collected. 3-oxoacyl reductase was modified in PyMol by aligning the protein structure 4I08 and adding NADPH to 2C07. Large scale protein expression was conducted on 10/19/2013. The cell lysate collected after spin down (JA10 rotor, 6000 rpm, 4degrees Celsius, 20 min) was transfered into a 15 ml conical tube and placed in the -80 freezer. 3.33 g of cell lysate was collected.
Fig: SDS Page gel of PfFabG
Lane 0: Elution 2Lane 1: Elution 1Lane 2: WashLane 3: Flow throughLane 4: Soluble FractionLane 5: Cell lysate after IPTG inductionLane 6: Cell lysate before IPTG inductionLane 7: ColorPlus Prestained Protein Ladder In the Elution 1 and 2 lane, there is a band at approximately 80 kDa. PfFabG has a molar mass of 76626.6 g/mol. This shows promise of a successful expression of PfFabG. However, there is large amounts of contamination and not strong band at the correct size. The intensity of the band contradicts the concentration determined by Nanodrop. FPLC will either be pursued or re-expression may be necessary.
Fig: Concentration analysis of PfFabG after spinning down in the concentrator.
Week 5 & 6: 09/23/2013 - 10/06/2013
Nice Job on your page. Try to elaborate a bit more on your analysis such as if you have collected sufficient data to move onto next steps. Thank you. -Max 10/07/2013
Summary of Week 5 & 6: The BL21 (DE3) cells expressing PfFabG were sonicated to lyse the cells and release the protein into solution. The cell lysate was then spun down at 20,000 g at 4 degrees Celsius for 30 minutes total. The supernatant was collected and prepared for protein purification. Elution 1 and 2 of protein purification was collected in two 15 mL conical tubes and a SDS-PAGE gel was conducted for protein characterization. The protein gel is not shown here though because Grace was nice enough to dry the gel for me since I'll be out of town.
Fig 34: Cell pellet after sonication and centrifugation. The cleared lysate was saved in a separate 15 mL conical tube, filtered (0.45 um), and stored in the -20 degree Celcius freezer.
Fig 35: Concentration analysis of PfFabG after purification - Elution 1
The average concentration of Elution 1 is 2.01mg/mL with an average 260/280 value of 1.895. It was determined that the concentration of eluted protein demonstrated a large yield.
Fig 40: Concentration analysis of PfFabG after purification - elution 2
Week 3 & 4: 09/09/2013 - 09/22/2013Melissa - good work and well layed out. Dr B 100113
Summary of Week 3 & 4: DNA sequencing request for Sample B2 was analyzed and it was confirmed that the plasmid was a positive clone of PfFabG. Plasmid was transformed in DH5alpha and BL21(DE3) cells. Transformation in DH5alpha cells are necessary to increase the stock plasmid available. First attempt was unsuccessful. Second attempt was successful only for the transformation of PfFabG in BL21(DE3) cells. BL21 (DE3) cells underwent large scale protein expression. The cells were centrifuged, the supernatant disposed of, and resuspended in lysis buffer. The sample was placed in the -80 degrees Celsius freezer.
Figure 29: Blast nucleotide analysis between CDS codon optimized sequence (Query) and the received DNA sequence from DNA sequencing (Subject). The blast analysis that is encompassed by the green boxes are highlighted to demonstrate the area of the blast nucleotide analysis that is 100% confident.
Figure 30: Blast nucleotide analysis between CDS codon optimized sequence (Query) and the received DNA sequence from DNA sequencing (Subject). The blast analysis that is encompassed by the green boxes are highlighted to demonstrate the area of the blast nucleotide analysis that is 100% confident.
Because the area that shows 100% confidence in both sequences overlap, Sample B2 can be concluded that it is indeed a positive clone of PfFabG. Table 1: Comparison table of the Blast nucleotide analysis of the samples submitted to DNA sequencing. The last column indicates whether or not the sample was a positive or negative clone.
Fig 31: Transformation plates of BL21(DE3) and Dh5alpha E. coli cells. Plates show unsuccesful transformation.
Fig 32: Transformation plates of Bl21(DE3) cells with PfFabG in pnic-Bsa4 plasmid. Transformation plates show successful transformation.
Fig 33: Transformation plates of PfFabG in DH5alpha E. coli cells. Plates show unsuccessful transformation.
Week 1 & 2: 8/28/2013 - 09/07/2013Melissa - do you have more wet lab work here? Dr. B 090913
Summary of Week 1 & 2: Blast analysis of the received DNA sequence from the Core Lab was conducted to determine if there was a match to a positive clone. Sample B2 showed 99% query coverage and 99% identities but the reverse pLIC sequenced request did not show as much success. The reverse and forward sequences were resubmitted for DNA sequencing.
Fig 28: Blast analysis of DNA sequencing request of Sample B2 using the pLIC forward primer. 99% query coverage was confirmed.
SUMMER 2013Week 8: 7/20/2013 - 7/27/2013
Summary of Week 8: After more pNIC-Bsa4 plasmid was recollected, pNIC was restriction enzyme digested using Bsa1 to prepare the plasmid as an accepting vector again. Cohesive end generation was reconducted. Essentially the first cloning attempt was reconducted except the ratios between the accepting vector and the cohesive inserts were modified.
Fig 27: Successful cloning attempt of PfFabG in Dh5alpha cells. The ratio of accepting vector to insert are indicated below each image.
Week 7: 7/15/2013 - 7/19/2013
Summary of Week 7: Pnic-Bsa4 plasmid was restriction enzyme digested using BsaI to prepare the plasmid as an accepting vector. Then the cohesive end generation was conducted on PCR inserts and the accepting vector. Then the inserts and vector were annealed and transformed in DH5alpha cells and placed on LB+Kan+Sucrose (5%) plates. The first cloning attempt was unsuccessful so more pnic-Bsa4 was grown overnight and midi-prepped. Because the concentration of the purified plasmid was so low, more pnic-Bsa4 will be grown and midiprepped.
Fig 26: Restriction enzyme digest analysis of pnic-Bsa4 using Bsa1. Standard used was 1 kb ladder (located in lane 3).
Fig 27: Unsuccessful cloning attempt of PfFabG transformed in pnic-Bsa4 in Dh5alpha cells.
Week 6: 7/8/2013 - 7/12/2013 Good work Melissa - Dr. B 071713
Summary of Week 6: PfDXR was FPLC filtrated to further purify the sample. The DNA sequence request for pnic-Bsa4 was received and the DNA sequence was analyzed. On 7/10/13, secondary PCR was re-conducted using a different annealing temperature of 66 degrees Celsius and a gel was run. After determining that secondary PCR was successful, PCR squared was conducted, yielding a total sample of 200 uL. Then the plasmid solution was cleaned up using the Sigma PCR Clean Up kit. After collecting the sample, the total average concentration of the solution was 160.95 ng/uL.
Fig 20: Gel analysis of FabG for P. falciparum that underwent secondary PCR using designed tail primers. T (located in lane 3). The 1 kb ladder was used as the standard.
Because a distinct band did not appear at the correct location (935 base pairs), secondary PCR was re-conducted.
Fig 21: Nucleotide blast analysis of pnic-Bsa4 for cloning. Query is pnic-Bsa4 sequence that was received from DNA sequencing lab.
Fig 22: Gel analysis of secondary PCR (redo 1) of FabG with tail primers (Lane 3). Standard used was a 1 kb ladder (Lane 2).
Fig 23: Gel analysis of PCR squared to confirm amplification of FabG (P. falciparum), located in lanes 5-8. Standard used was 1 kb ladder.
Fig 25: Nanodrop concentration analysis of FabG (P. falciparum) after PCR cleanup.
Week 5: 7/1/2013 - 7/5/2013 Summary of Week 5: The tail primers for FabG for P. falciparum were designed and ordered on 7/1/2013. On Tuesday, 7/2/2013, the primers were diluted from 100 uM to 1 uM in the master mix of the 24 oligonucleotides necessary to synthesize the gene. Overnight cultures of pnic-Bsa4 in DH5alpha cells were grown and midiprepped. On 7/3/2013, primary PCR on the oligo mix was conducted. Because the tail primers were not available on Wednesday, secondary PCR was conducted using the first and last oligonucleotide as the primers. The identity of the primary and secondary PCR were confirmed using an agarose gel (shown in Fig 18). On 7/5/2013, PCR samples were prepared for secondary PCR using tail primers designed on 7/1/2013.
Fig 18: Gel analysis of primary and secondary PCR on FabG in Plasmodium falciparum (located on the left side of the gel - lanes 1-3)
Lane 1: 1 kb ladderLane 2: Primary PCR sample for FabGLane 3: Secondary PCR sample for FabGLane 4: N/ALane 5-7: Oscar V.'s samples
Fig 19: Concentration analysis of pnic Bsa4 with an average concentration of 49.3 ng/uL, average 260/280 value of 1.96 and average 260/230 value of 8.88
WEEK 4: 6/24/2013 - 6/28/2013 Summary of Week 4: A homemade SDS-PAGE gel was made in lab on 6/25/2013. Then on 6/26/2013, the sample of PfDXR in BL21(DE3) cells were sonicated to release the protein from the cell and then the protein was purified using a Ni-NTA column. Samples were collected from each step (from cell lysate to Elution 2) and were characterized using the SDS-PAGE gel made. On 6/28/2013, a sample of pGBR22 underwent an restriction enzyme digest.
Fig 14: Concentration analysis at 280 nm of PfDXR Elution 1 after purification
Fig 15: Concentration analysis at 280 nm of PfDXR Elution 2 after purification
Fig 16: SDS-PAGE gel of PfDXR
Lane 1: N/ALane 2: PAGE gel rulerLane 3: Cell lysate before inductionLane 4: Cell lysate after inductionLane 5: Soluble fractionLane 6: Flow throughLane 7: WashLane 8: Elution 1Lane 9: Elution 2Lane 10: N/A
Fig 17: Gel analysis of pGBR22 restriction enzyme digest. Left side - Melissa H. Right side - Antonio G.
Lane 1: 1 kb DNA ladderLane 2: Uncut plasmidLane 3: EcoRI - HFLane 4: PvuIILane 5: EcoRI-HF + PvuII
WEEK 3: 6/17/2013 - 6/21/2013 Summary of Week 3: The DNA sequence submitted to the DNA sequencing core during Week 2 for FrTUHP in pnic-Bsa4 was analyzed in order to confirm the presence of the correct plasmid. On 6/17/2013, a gel was run on the GFP PCR conducted on 6/13/2013 to confirm the identity of the plasmid. on 6/18/2013, a PCR was run on pnic-Bsa4 and a gel was run. A large scale protein expression of PfDXR in pnic-Bsa4 in BL21(DE3) E. coli cells. was conducted on 6/18/2013. A growth curve was calculated in order to measure the concentration of the bacteria in media up to 0.5 OD and then 263 uL IPTG was added. The 3.37 g of cells were harvested by centrifugation, re-suspended in re-suspension buffer, and then stored at -80 degree Celsius. pNIC-Bsa4 was amplified using PCR on 6/19/2013 but when the gel was run on 6/20/2013, no DNA was present and thus the PCR was uneffective.
Fig 10: DNA sequence received for FrTUHP in pnic-Bsa4
Fig 11: Blast nucleotide sequence confirming the identity of FrTUHP in pnic-Bsa4 . Query is DNA sequence received from lab and subject is actual DNA sequence
The source of the nucleotide sequence used in the comparison was Joey and Christina's primer design sequence used in their research last summer. Their nucleotide sequence was optimized to for E. coli so the sequences in the NCBI database were not appropriate for comparison.
Fig 12: Gel analysis of GFP
A gel was run on GFP PCR that was conducted last Thursday, 6/13/2013.
The samples in each lane are as followed: 1: 100 bp DNA ladder2: 0.011 ng/ul GFP with VDS forward and reverse primers3: 0.11 ng/ul GFP with VDS forward and reverse primers4: 1.1 ng/ul GFP with VDS forward and reverse primers5: NO GFP with VDS forward and reverse primers6: 100 bp DNA ladder7: 0.011 ng/ul GFP with M13 forward and reverse primers8: 0.11 ng/ul GFP with M13 forward and reverse primers9: 1.1 ng/ul GFP with M13 forward and reverse primers10: ??? 11.1 ng/ul GFP with M13 forward and reverse primers
In the protocol provided for this PCR run, there was supposed to be no DNA present in Lane 10 which would be used as a control. Because there is a band for lane 10 and the band is more florescent than Lane 7-9, it is possible that the 11.1 ng/uL (1:100 dilution) was placed in the sample.
Fig 13: Gel analysis of pnic Bsa4 on left
WEEK 2: 6/10/2013 - 6/14/2013 Melissa - excellent work! Specify which side of the PCR gel is yours. -- Dr. B
Fig 7: BlastP comparison of pGBR plasmid DNA sequence to confirm identity of pGBR22 plasmid, showing 100% coverage on 100% identities
Fig 8: Concentration determination of FrTUHP in pnic-Bsa4 at 260 nm - Measurement 1
Fig 9: Concentration determination of FrTUHP in pnic-Bsa4 at 260 nm - Measurement 2
Average concentration was 70.1 ng/uL.
WEEK 1: 6/3/2013 - 6/7/2013
Fig 1: First nanodrop analysis of pGBR22 (purple? protein) in order to determine the purity and the concentration of the DNA plasmid
Fig 1: Second nanodrop analysis of pGBR22 (purple? protein) in order to determine the purity and the concentration of the DNA plasmid
The average concentration of the pGBR22 plasmid was 185.05 ng/ul. The average ratio between the purity in relation to the protein is 1.9. The average ratio between the purity in relation to other contaminants is 2.665. The average absorbance at 230 nm is 1.391. The purity of the protein in comparison to other contaminants is low because the ideal ratio value is 2.1 but the ratio determined was 2.665.
Fig 3: E. Coli (DHS-alpha) bacterial culture on LB and Kanamycin plate with 1 ng FrTUHP in pnic-Bsa4 after one day in 37 degree C incubator
Fig 4: E. Coli (DHS-alpha) bacterial culture with 5 ng FrTUHP in pnic-Bsa4 on LB and Kanamycin plate after one day in 37 degree C incubator
Fig 5: E. Coli (DHS-alpha) bacterial culture with 25 ng FrTUHP in pnic-Bsa4 on LB and Kanamycin plate after one day in 37 degree C incubator
Because the plasmid is a low count plasmid, the number of colonies on the plate were much lower than was to be expected. Because E. coli competes for space, it was expected that the higher the concentration of DNA plasmid, the less amount of E. coli colonies per plate.
FALL 2013Week 13 & 14: 11/18/2013 - 12/1/2013
Summary of Week 13 & 14: Another sample of PfFabG was expressed, purified, and characterized. However, when the SDS-Page gel was analyzed, there was little to no band at the correct location (~33 kDa) on the gel which means that expression was likely unsuccessful. More virtual screening was conducted and the data was combined together in an excel document.
When the samples (Elution 1, Elution 2, Concentrated Sample) were analyzed using the Nanodrop, the conentrations determined were very low. This provides more evidence that the protein did not expressed or was in the -80 degree celsius freezer for too long to be viable.
From left to right:
ColorPlus Protein Ladder
0: Before IPTG Induction
2: Soluble Fraction
3: Flowthrough
4: Wash
5: Elution 1
6: Elution 2
PfFabG is supposed to elute around the 33 kDa mark. From the gel you can see that the protein appears up until the wash sample and is missing in Elution 1 and 2. Based on the previous gel, I do not believe that there is a problem with the His-tag adhering to the Ni ions because there was a band at 33kDa in Elution 1 and 2 in the previous gel. There may have been a problem with purification in general. I used the same purification column as before so there could be an issue with the the age of the column or the incubation step.
Week 11 & 12: 11/4/13 - 11/17/2013
Good--nice job on the protein -UMSummary of Week 11 & 12: GOLD docking of the CB306 library was conducted and analyzed. An enzyme assay was conducted on the enzyme collected two weeks ago to test for enzyme functionality. Due to the results collected from the time based trial, it appears that the protein is indeed functional.
Table 1: Gold docking analysis of CB306 compound library during the initial screening. The first 33 compounds will be further analyzed for more thorough analysis.
Table 2: Gold docking scores of the second run of the top 33 compounds in the CB306 library. The top four scores/10% of the second run are shown.
During the second run of the CB306 docking, the top ligands that were determined in the previous Run 2 were not ranked as highly in the second run. There is however a low standard deviation between each score. Although the scores may have ranked differently, the GOLD score between each ligand does not change by a large percentage.
Because there is a subtle decrease in the absorbance of NADPH at 340 nm, this may indicate that the protein is functional. For the light blue line, the peaks that appear in the line are times when more enyme was added to the measured sample.
Week 9 & 10: 10/23/13 - 11/3/2013
Good work Melissa! Do you have your FPLC results that you could post on here? Also, it would be good to include analysis on your virtual screening results. - Suman 11/4/13
Summary of Week 9 & 10: Protein was expressed, purified, and characterized during week 9. The protein was loaded into the FPLC for further concentration. Then the protein
was stored in 20% glycerol. Protein expression was re-conducted due to a low concentration in protein. For virtual screening, 2C07 was analyzed through MolProbity and control and CB306 docking was conducted. Then the DH5alpha cells from cloning during the summer were regrown on a master plate and midiprepped for a larger stock of plasmid concentration.
Fig: Concentration analysis of PfFabG after protein purification. The average concentration after Elution 1 was 0.645 mg/mL.
Fig: Concentration analysis of PfFabG after FPLC but before concentrating the sample. The average concentration is very low at 0.155 mg/mL.
Overall, protein purification will have to be scaled up in order to collect a larger yield of protein during round 3 of expression.
Molprobity AnalysisResolution: 1.5 angstromsClashscore: 11.95Poor rotamers: 0Ramachandran outliers: 0Ramachandran favored: 236Cbeta deviations >0.25 A: 0MolProbity score ^: 1.69Residues with bad bonds: 0/982Residues with bad angles: 1/1224Error in Active Site: Contacts, McSc contacts, Hets contacts, 1 angle
Overall there are few errors in active site and the protein structure was modified with flips and without hydrogens.
Table 1: Gold score table of control docking of PfFabG
Week 7 & 8: 10/07/2013 - 10/20/13 Good work Melissa. Try to include a brief analysis of the Nanodrop data. Do you have any virtual data? Thank you. -Max 10/21/13
Summary of Week 7 & 8: SDS-PAGE gel was analysed. PfFabG was spun down in the concentrator, yielding an average concentration of 2.52 mg/mL. Protein expression was attempted but due to time constraints, it could not be achieved. More preparation towards virtual screening was conducted. The positive and negative control ligands were collected. 3-oxoacyl reductase was modified in PyMol by aligning the protein structure 4I08 and adding NADPH to 2C07. Large scale protein expression was conducted on 10/19/2013. The cell lysate collected after spin down (JA10 rotor, 6000 rpm, 4degrees Celsius, 20 min) was transfered into a 15 ml conical tube and placed in the -80 freezer. 3.33 g of cell lysate was collected.
Lane 0: Elution 2Lane 1: Elution 1Lane 2: WashLane 3: Flow throughLane 4: Soluble FractionLane 5: Cell lysate after IPTG inductionLane 6: Cell lysate before IPTG inductionLane 7: ColorPlus Prestained Protein Ladder
In the Elution 1 and 2 lane, there is a band at approximately 80 kDa. PfFabG has a molar mass of 76626.6 g/mol. This shows promise of a successful expression of PfFabG. However, there is large amounts of contamination and not strong band at the correct size. The intensity of the band contradicts the concentration determined by Nanodrop. FPLC will either be pursued or re-expression may be necessary.
Week 5 & 6: 09/23/2013 - 10/06/2013
Nice Job on your page. Try to elaborate a bit more on your analysis such as if you have collected sufficient data to move onto next steps. Thank you. -Max 10/07/2013
Summary of Week 5 & 6: The BL21 (DE3) cells expressing PfFabG were sonicated to lyse the cells and release the protein into solution. The cell lysate was then spun down at 20,000 g at 4 degrees Celsius for 30 minutes total. The supernatant was collected and prepared for protein purification. Elution 1 and 2 of protein purification was collected in two 15 mL conical tubes and a SDS-PAGE gel was conducted for protein characterization. The protein gel is not shown here though because Grace was nice enough to dry the gel for me since I'll be out of town.
The average concentration of Elution 1 is 2.01mg/mL with an average 260/280 value of 1.895.
It was determined that the concentration of eluted protein demonstrated a large yield.
Week 3 & 4: 09/09/2013 - 09/22/2013Melissa - good work and well layed out. Dr B 100113
Summary of Week 3 & 4: DNA sequencing request for Sample B2 was analyzed and it was confirmed that the plasmid was a positive clone of PfFabG. Plasmid was transformed in DH5alpha and BL21(DE3) cells. Transformation in DH5alpha cells are necessary to increase the stock plasmid available. First attempt was unsuccessful. Second attempt was successful only for the transformation of PfFabG in BL21(DE3) cells. BL21 (DE3) cells underwent large scale protein expression. The cells were centrifuged, the supernatant disposed of, and resuspended in lysis buffer. The sample was placed in the -80 degrees Celsius freezer.
Because the area that shows 100% confidence in both sequences overlap, Sample B2 can be concluded that it is indeed a positive clone of PfFabG.
Table 1: Comparison table of the Blast nucleotide analysis of the samples submitted to DNA sequencing. The last column indicates whether or not the sample was a positive or negative clone.
Week 1 & 2: 8/28/2013 - 09/07/2013Melissa - do you have more wet lab work here? Dr. B 090913
Summary of Week 1 & 2: Blast analysis of the received DNA sequence from the Core Lab was conducted to determine if there was a match to a positive clone. Sample B2 showed 99% query coverage and 99% identities but the reverse pLIC sequenced request did not show as much success. The reverse and forward sequences were resubmitted for DNA sequencing.
SUMMER 2013Week 8: 7/20/2013 - 7/27/2013
Summary of Week 8: After more pNIC-Bsa4 plasmid was recollected, pNIC was restriction enzyme digested using Bsa1 to prepare the plasmid as an accepting vector again. Cohesive end generation was reconducted. Essentially the first cloning attempt was reconducted except the ratios between the accepting vector and the cohesive inserts were modified.
Week 7: 7/15/2013 - 7/19/2013
Summary of Week 7: Pnic-Bsa4 plasmid was restriction enzyme digested using BsaI to prepare the plasmid as an accepting vector. Then the cohesive end generation was conducted on PCR inserts and the accepting vector. Then the inserts and vector were annealed and transformed in DH5alpha cells and placed on LB+Kan+Sucrose (5%) plates. The first cloning attempt was unsuccessful so more pnic-Bsa4 was grown overnight and midi-prepped. Because the concentration of the purified plasmid was so low, more pnic-Bsa4 will be grown and midiprepped.
Week 6: 7/8/2013 - 7/12/2013
Good work Melissa - Dr. B 071713
Summary of Week 6: PfDXR was FPLC filtrated to further purify the sample. The DNA sequence request for pnic-Bsa4 was received and the DNA sequence was analyzed. On 7/10/13, secondary PCR was re-conducted using a different annealing temperature of 66 degrees Celsius and a gel was run. After determining that secondary PCR was successful, PCR squared was conducted, yielding a total sample of 200 uL. Then the plasmid solution was cleaned up using the Sigma PCR Clean Up kit. After collecting the sample, the total average concentration of the solution was 160.95 ng/uL.
Because a distinct band did not appear at the correct location (935 base pairs), secondary PCR was re-conducted.
Week 5: 7/1/2013 - 7/5/2013
Summary of Week 5: The tail primers for FabG for P. falciparum were designed and ordered on 7/1/2013. On Tuesday, 7/2/2013, the primers were diluted from 100 uM to 1 uM in the master mix of the 24 oligonucleotides necessary to synthesize the gene. Overnight cultures of pnic-Bsa4 in DH5alpha cells were grown and midiprepped. On 7/3/2013, primary PCR on the oligo mix was conducted. Because the tail primers were not available on Wednesday, secondary PCR was conducted using the first and last oligonucleotide as the primers. The identity of the primary and secondary PCR were confirmed using an agarose gel (shown in Fig 18). On 7/5/2013, PCR samples were prepared for secondary PCR using tail primers designed on 7/1/2013.
WEEK 4: 6/24/2013 - 6/28/2013
Summary of Week 4: A homemade SDS-PAGE gel was made in lab on 6/25/2013. Then on 6/26/2013, the sample of PfDXR in BL21(DE3) cells were sonicated to release the protein from the cell and then the protein was purified using a Ni-NTA column. Samples were collected from each step (from cell lysate to Elution 2) and were characterized using the SDS-PAGE gel made. On 6/28/2013, a sample of pGBR22 underwent an restriction enzyme digest.
Lane 1: N/ALane 2: PAGE gel rulerLane 3: Cell lysate before inductionLane 4: Cell lysate after inductionLane 5: Soluble fractionLane 6: Flow throughLane 7: WashLane 8: Elution 1Lane 9: Elution 2Lane 10: N/A
Lane 1: 1 kb DNA ladderLane 2: Uncut plasmidLane 3: EcoRI - HFLane 4: PvuIILane 5: EcoRI-HF + PvuII
WEEK 3: 6/17/2013 - 6/21/2013
Summary of Week 3: The DNA sequence submitted to the DNA sequencing core during Week 2 for FrTUHP in pnic-Bsa4 was analyzed in order to confirm the presence of the correct plasmid. On 6/17/2013, a gel was run on the GFP PCR conducted on 6/13/2013 to confirm the identity of the plasmid. on 6/18/2013, a PCR was run on pnic-Bsa4 and a gel was run. A large scale protein expression of PfDXR in pnic-Bsa4 in BL21(DE3) E. coli cells. was conducted on 6/18/2013. A growth curve was calculated in order to measure the concentration of the bacteria in media up to 0.5 OD and then 263 uL IPTG was added. The 3.37 g of cells were harvested by centrifugation, re-suspended in re-suspension buffer, and then stored at -80 degree Celsius. pNIC-Bsa4 was amplified using PCR on 6/19/2013 but when the gel was run on 6/20/2013, no DNA was present and thus the PCR was uneffective.
The source of the nucleotide sequence used in the comparison was Joey and Christina's primer design sequence used in their research last summer. Their nucleotide sequence was optimized to for E. coli so the sequences in the NCBI database were not appropriate for comparison.
A gel was run on GFP PCR that was conducted last Thursday, 6/13/2013.
The samples in each lane are as followed:
1: 100 bp DNA ladder2: 0.011 ng/ul GFP with VDS forward and reverse primers3: 0.11 ng/ul GFP with VDS forward and reverse primers4: 1.1 ng/ul GFP with VDS forward and reverse primers5: NO GFP with VDS forward and reverse primers6: 100 bp DNA ladder7: 0.011 ng/ul GFP with M13 forward and reverse primers8: 0.11 ng/ul GFP with M13 forward and reverse primers9: 1.1 ng/ul GFP with M13 forward and reverse primers10: ??? 11.1 ng/ul GFP with M13 forward and reverse primers
In the protocol provided for this PCR run, there was supposed to be no DNA present in Lane 10 which would be used as a control. Because there is a band for lane 10 and the band is more florescent than Lane 7-9, it is possible that the 11.1 ng/uL (1:100 dilution) was placed in the sample.
WEEK 2: 6/10/2013 - 6/14/2013
Melissa - excellent work! Specify which side of the PCR gel is yours. -- Dr. B
CCCATATGGTCGACCTGCAGGCGGCCGCACTAGTGATTTTGATTGATTGAAGGAGAAATAT
CATGAGTGTGATCGCTAAACAAATGACCTACAAGGTTTATATGTCAGGCACGGTCAATGGA
CACTACTTTGAGGTCGAAGGCGATGGAAAAGGAAAGCCTTACGAGGGGGAGCAGACGGT
AAAGCTCACTGTCACCAAGGGTGGACCTCTGCCATTTGCTTGGGATATTTTATCACCACTGT
CTCAATACGGAAGCATACCATTCACCAAGTACCCTGAAGACATCCCTGATTATGTAAAGCA
GTCATTCCCTGAGGGATATACATGGGAGAGGATCATGAACTTTGAAGATGGTGCAGTGTGT
ACTGTCAGCAATGATTCCAGCATCCAAGGCAACTGTTTCATCTACAATGTCAAAATCTCTGG
TGTGAACTTTCCTCCCAATGGACCTGTTATGCAGAAGAAGACACAGGGCTGGGAACCCAAC
ACTGAGCGTCTCTTTGCACGAGATGGAATGCTGATAGGAAACAACTTTATGGCTCTGAAGTT
GGAAGGAGGTGGTTACTATTTGTGTGAATTCAAATCTACTTACAAGGCAAAGAAGCCTGTGA
GGATGCCAGGGTATCACTATGTTGACCGCAAACTGGATGTAACCAGTCACAACAAGGATTA
CACATTTGTTGAGCAGTGTGAAATATCCATTGCACGCCACTCTTTGCTCGGTCATCACCATCA
CCATCACTAAAATCCCGCGGCCATGGCGGCCGGGAGCATGCGACGTCGGGCCCAATTCGC
CCTATAGTGAGTCGTATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC
CTGGNGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGNGTAATAGC
GAANAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGNGAATGGNCN
NNNCCTGTAGCGGCGCATTAAGCGCGGCNGGGNGNGGNNGNTACNNNCAGCGNGACNGC
NNNCNCTTNCNGCNCCNANCGNCCGCTCNTTNNNNTTCNTNCNNNNTTCTNNCNNCNTNNNN
NNTTNCCNNNAGNNNTAANNNGGGNNCCNTTNNNNNCNANTTNNNNTTNNGNNNCNNNNNN
NNNNNNNANNNNNNANNNNNNNNNNGGNNTNNCCNNNNNNANNNNNNNNTNNNNNNCCN
Average concentration was 70.1 ng/uL.
WEEK 1: 6/3/2013 - 6/7/2013
The average concentration of the pGBR22 plasmid was 185.05 ng/ul.
The average ratio between the purity in relation to the protein is 1.9.
The average ratio between the purity in relation to other contaminants is 2.665.
The average absorbance at 230 nm is 1.391.
The purity of the protein in comparison to other contaminants is low because the ideal ratio value is 2.1 but the ratio determined was 2.665.
Because the plasmid is a low count plasmid, the number of colonies on the plate were much lower than was to be expected. Because E. coli competes for space, it was expected that the higher the concentration of DNA plasmid, the less amount of E. coli colonies per plate.